Critical Limb Ischemia (CLI) is a common problem that often results in major limb amputation. Circulating endothelial progenitor cells likely play a role in the pathogenesis of CLI;the number and function of these cells that are mobilized from the bone marrow are reduced in the elderly and diabetics, groups at high risk for CLI. Allogenic Umbilical Cord Blood (UCB) derived CD133+ hemangioblasts demonstrate greater activity for stimulating angiogenesis. However, there are no clinical trials of UCB-derived cell lines for CLI and the number of cells available from a cord sample is limited. The long-term objective is to develop a clinically relevant approach, through nanomaterials engineering, to efficiently expand human allogenic UCB derived CD133+ hemangioblasts and to demonstrate the biologic and therapeutic feasibility to treat CLI. In this proposed study, Specific Aim I will optimize clinical grade nanofiber scaffold ex vivo expansion of UCB derived CD133+ hemangioblasts from fresh and cryopreserved UCBs in a Good Manufacturing Practice (GMP) based cell culture and production facility. Specific Aim IIa will test survival of the expanded UCB allograft in a Histocompatibility Leukocyte Antigen (HLA)-matched, unrelated, non-immunosuppressed recipients. To test this aim 5 women with CLI will undergo implantation of HLA matched, gender mismatched UCB-derived cells subcutaneously with biopsy of the delivery site used to determine cell survival. To identify transplanted cells the specimens will be examined for viable Y-chromosome bearing cells using the Fluorescent in situ hybridization techniques (FISH). Specific Aim IIb will establish the therapeutic potential of the expanded HLA matched UCB allograft as a strategy to improve tissue perfusion in CLI. To test this aim 10 patients with CLI will receive HLA matched, ex vivo expanded, UCB-derived cells injected into the affected limb. This Aim will have 80% power to detect a 10 mmHg improvement in Transcutaneous Oxygen Pressure (TcPO2). PUBLIC HEALTH RELEVANCE: The overall objective of this project is to demonstrate the safety and efficacy of injecting ex vivo expanded allogeneic (from other people) umbilical cord blood-derived stem cells (CD133+ cells) for the treatment of critical limb ischemia (CLI). By providing enough blood supply to the ischemic limb, Arteriocyte hopes to observe reduced rest pain, increased exercise capacity, increased skin surface oxygen pressure and improved ulcer healing in the CLI patients. Arteriocyte believes through its stem cell therapy, patients with CLI will benefit from functional improvement, lower amputation risks, and save significant healthcare costs associated with CLI.